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FastCAT accelerates absolute quantification of proteins by using multiple short non-purified chimeric standards

ABSTRACT: Absolute (molar) quantification of proteins provides the analytical rationale for system-level modelling of diverse molecular mechanisms. FastCAT method employs multiple short (<50 kDa) stable-isotope labeled chimeric proteins (CPs) composed of concatenated quantotypic (Q-) peptides representing the quantified proteins. Each CP also comprises scrambled sequences of reference (R-) peptides that relate its abundance to a single protein standard (BSA). FastCAT not only alleviates the need in purifying CP or using SDS-PAGE, but also improves the accuracy, precision and dynamic range of the absolute quantifications by grouping Q-peptides according to the expected abundance of target proteins. We benchmarked FastCAT against the reference method of MS Western and tested it in the direct molar quantifications of neurological markers in human cerebrospinal fluid at the low ng/mL level.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cerebrospinal Fluid

SUBMITTER: Ignacy Rzagalinski

LAB HEAD: Andrej Shevchenko

PROVIDER: PXD030728 | Pride | 2022-04-01

REPOSITORIES: Pride

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FastCAT accelerates absolute quantification of proteins by using multiple short non-purified chimeric standards

Project description:Absolute (molar) quantification of proteins provides the analytical rationale for system-level modelling of diverse molecular mechanisms. FastCAT method employs multiple short (<50 kDa) stable-isotope labeled chimeric proteins (CPs) composed of concatenated quantotypic (Q-)peptides representing the quantified proteins. Each CP also comprises scrambled sequences of reference (R-)peptides that relate its abundance to a single protein standard (BSA). FastCAT not only alleviates the need in purifying CP or using SDS-PAGE, but also improves the accuracy, precision and dynamic range of the absolute quantifications by grouping Q-peptides according to the expected abundance of target proteins. We benchmarked FastCAT against the reference method of MS Western and tested it in the direct molar quantifications of neurological markers in human cerebrospinal fluid at the low ng/mL level.

2022-04-04 | PXD030762 | panorama

Drosophila melanogaster eye Proteomics

Project description:Absolute (molar) quantification determines proteins stoichiometry in complexes, networks and metabolic pathways. We employed MS Western workflow to determine molar abundances of proteins critical for morphogenesis and phototransduction (PT) in eyes of Drosophila melanogaster using a single chimeric 264 kDa protein standard that covers, in total, 197 peptides from 43 proteins. Each protein was independently quantified with 2 to 4 proteotypic peptides with the coefficient of variation of less than 15 %, better than 1000-fold dynamic range and sub-femtomole sensitivity. We determined molar abundances and stoichiometric ratios of the components of the PT machinery and the rhabdomere, and how they are changing when rhabdomere morphogenesis is perturbed by genetic manipulation of the evolutionary conserved gene crumbs (crb).

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The hematopoietic stem cell in chronic phase CML is characterized by a transcriptional profile resembling normal myeloid progenitor cells and reflecting loss of quiescence

Project description:We found that composition of cell subsets within the CD34+ cell population is markedly altered in chronic phase (CP) chronic myeloid leukemia (CML). Specifically, proportions and absolute cell counts of common myeloid progenitors (CMP) and megakaryocyte-erythrocyte progenitors (MEP) are significantly greater in comparison to normal bone marrow whereas absolute numbers of hematopoietic stem cells (HSC) are equal. To understand the basis for this, we performed gene expression profiling (Affymetrix HU-133A 2.0) of the distinct CD34+ cell subsets from six patients with CP CML and five healthy donors. Euclidean distance analysis revealed a remarkable transcriptional similarity between the CML patients' HSC and normal progenitors, especially CMP. CP CML HSC were transcriptionally more similar to their progeny than normal HSC to theirs, suggesting a more mature phenotype. Hence, the greatest differences between CP CML patients and normal donors were apparent in HSC including downregulation of genes encoding adhesion molecules, transcription factors, regulators of stem-cell fate and inhibitors of cell proliferation in CP CML. Impaired adhesive and migratory capacities were functionally corroborated by fibronectin detachment analysis and transwell assays, respectively. Based on our findings we propose a loss of quiescence of the CML HSC on detachment from the niche leading to expansion of myeloid progenitors.

2008-12-01 | GSE11889 | GEO

Median Based Absolute Quantification using Fully Unlabelled Generic Internal Standard

Project description:By reporting molar abundances of proteins, absolute quantification determines their stoichiometry in complexes, pathways or networks and also relates them to abundances of non-protein biomolecules. Typically, absolute quantification relies either on protein- specific isotopically labelled peptide standards or on a semi-empirical calibration against the average abundance of peptides chosen from arbitrary selected standard proteins. Here we developed a generic protein standard FUGIS (Fully unlabelled Generic Internal Standard) that requires no isotopic labelling, synthesis of standards or external calibration and is applicable to proteins of any organismal origin. FUGIS is co-digested with analysed proteins and enables their absolute quantification in the same LC-MS/MS run. By using FUGIS, median based absolute quantification (MBAQ) workflow provides similar quantification accuracy compared to isotopically-labelled peptide standards and outperforms methods based on external calibration or selection of best ionized reporter peptides (Top3 quantification) with a median quantification error less than 15%

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Supplementation of specific collagen peptides following high-load resistance exercise upregulates gene expression in pathways involved in skeletal muscle signal transduction

Project description:Previous evidence suggests that resistance training in combination with specific collagen peptides (CP) improves adaptive responses of the muscular apparatus. Although beneficial effects have been repeatedly demonstrated, the underlying mechanisms are not well understood. Therefore, the primary objective of the present randomized trial was to elucidate differences in gene expression pathways related to myofibrillar protein synthesis following acute high-load resistance exercise with and without CP intake. Recreationally active male participants (n=30) were equally randomized to high-load (80% one-repetition maximum) leg extension exercise in combination with 15g CP or placebo (PLA) supplementation. Muscle biopsies from the vastus lateralis muscle were obtained at baseline as well as 1h, 4h and 24h post exercise to investigate gene expression using next generation sequencing analysis. Several important anabolic pathways including PI3K-Akt and MAPK pathways were significantly upregulated at 1h and 4h post-exercise (p<0.05). Significant between-group differences for both pathways were identified at the 4h time point. Gene expression related to the mTOR pathway demonstrated a higher visual increase in the CP group compared to PLA with no statistically significant group differences. The current findings revealed a significantly higher upregulation of key anabolic pathways (PI3K-Akt, MAPK) in human skeletal muscle in response to acute resistance training combined with intake of 15 g collagen peptides compared to placebo. Further investigations are needed to examine potential long-term effects on anabolic pathways on the protein level.

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Homeostatic Adjustment and Metabolic Remodeling in Glucose-limited Yeast Cultures Time Course 2

Project description:Time course of batch growth. Reference (channel 1) was a culture grown in MD medium with 2.4 g/L glucose, with 5 slpm air-flow, stirring at 400 rpm and a constant 300C temperature, dilution 0.25 volumes/hour. The experimental (channel 2) samples were grown in batch for the indicated time. Groups of assays that are related as part of a time series. FactorCategory: Elapsed Time; name: Elapsed Time; Measurement: time(absolute) 6 h; name: 34357_Elapsed Time; Measurement: time(absolute) 10.5 h; name: 34365_Elapsed Time; Measurement: time(absolute) 8 h; name: 34358_Elapsed Time; Measurement: time(absolute) 9 h; name: 34359_Elapsed Time; Measurement: time(absolute) 11 h; name: 34368_Elapsed Time; Measurement: time(absolute) 9.5 h; name: 34360_Elapsed Time; Measurement: time(absolute) 10 h; name: 34362_Elapsed Time Keywords: time_series_design

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Project description:The dataset aims at comparing gene expression in the developing lower and upper first molars of two mouse strains: DUHi and FVB. We studied molar tooth germ an early cap stage, looking for differences in activation-inhibition mechanisms that promote tooth formation. 19202 genes are in common between FVB and reference strain C57B6 and have a MGI annotation. Out of these genes, 2234 genes (11.6%) presented a significant difference of expression between the mapping on the reference strain C57B6 and the mapping on FVB strain (DESeq2, adjusted P value < 0.05, considering the mapping effect that is with 12 replicates). This is presumably a mapping artifact, due to the sequence divergence between mouse strains. These genes were removed, and the remaining 16,968 genes were retained for further analysis. 3619 genes were found to be differentially expressed between the two strains in the lower and upper molar (jaw+strain)

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Novel antioxidative peptides with potential to reduce psoriatic changes in PBMCs

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